While Mesenchymal Stem Cell (MSC)-based regenerative therapies have shown promise for treating osteoarthritis (OA), the underlying mechanisms remain unclear. Remarkably, recent evidence suggests MSCs can rescue injured cells by donating functional mitochondria (MT). However, intercellular MT transfer has not previously been reported in cartilage. While the mechanisms mediating MT transfer remain largely unknown, MT dysfunction in recipient cells is a prerequisite. We reported that MT dysfunction is one of the earliest responses of chondrocytes to injury, likely mediating downstream catabolic signaling cascades that drive OA. Our preliminary data provides the first evidence that bone marrow derived MSCs donate MT to chondrocytes undergoing MT dysfunction in both stained equine cells and murine cells expressing endogenous fluorophores. Further, pharmacological gap junction inhibition prevented MT transfer. Our broad goal is to investigate cellular mechanisms underlying MSC-chondrocyte MT transfer, and ultimately exploit these mechanisms to engineer new orthopedic regenerative therapies. Our strategy is to manipulate connexins, a class of gap junction proteins critical in intercellular communication. Connexin-43 (CX43) is a promising target for therapeutic development because it has been implicated in MT transfer in several cell types, and chondrocyte CX43 is strongly upregulated in OA. Therefore, the aims of this proposal are to investigate the role of CX43 in mediating physical interactions between MSCs and chondrocytes by (i) determining the relationship between CX43 expression and MT transfer, and (ii) designing and optimizing an optically transparent 3D co-culture device for direct control over the nature of intercellular interactions to identify how CX43 is involved in MT transfer. Our hypothesis is that increased CX43 expression in MSCs will lead to an increased frequency of MSC-chondrocyte MT transfer